fern cpdna====one particular taxon selaginella was problematic

8/7/2019 Fern CpDNA====One Particular Taxon Selaginella Was Problematic

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Molecular Phylogenetics and Evolution 36 (2005) 509522

www.elsevier.com/locate/ympev

1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ympev.2005.04.018

AmpliWcation of noncoding chloroplast DNA for phylogeneticstudies in lycophytes and monilophytes with a comparative example

of relative phylogenetic utility from Ophioglossaceae

Randall L. Small a,, Edgar B. Lickey a, Joey Shaw a, Warren D. Hauk b

a Department of Ecology and Evolutionary Biology, The University of Tennessee, Knoxville, TN 37996, USAb Department of Biology, Denison University, Granville, OH 43023, USA

Received 14 June 2004; revised 6 April 2005

Available online 1 June 2005

Abstract

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a signiWcant source of characters

for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and

Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte

and monilophyte species to determine the potential utility of PCR ampliWcation primers for 18 noncoding chloroplast DNA regions

that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying

lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of

noncoding chloroplast DNA, we analyzed the relative phylogenetic utility ofWve cpDNA regions for resolving relationships in Bot-

rychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnLUAAtrnFGAA intergenic spacer in this

group. To these published data we added sequences of the trnSGCUtrnGUUC intergenic spacer + the trnGUUC intron region, the

trnSGGArpS4 intergenic spacer + rpS4 gene, and the rpL16intron. Both the trnSGCUtrnGUUC and rpL16regions are highly variable

in angiosperms and the trnSGGArpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was

equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the Wve sampled regions the

trnSGCUtrnGUUC spacer + trnGUUC intron region provided the strongest support for the inferred phylogeny.

2005 Elsevier Inc. All rights reserved.

Keywords: Botrychium; Chloroplast DNA; Ferns; Lycophytes; Ophioglossaceae; Pteridophytes; Monilophytes

1. Introduction

Chloroplast DNA (cpDNA) sequences are the pri-

mary source of characters for phylogenetic studies inplants. Many early studies focused on protein-coding

gene sequences such as rbcL and were designed to eluci-

date phylogenetic relationships among higher-level taxa

(e.g., Chase et al., 1993). Subsequently, the potential util-

ity of noncoding regions of the chloroplast genome was

recognized for lower-level (intergeneric, interspeciWc,

and intraspeciWc) studies (e.g., Taberlet et al., 1991).

Noncoding regions such as introns and intergenic spac-

ers often display more variation on a per site basis than

coding regions, presumably due to fewer functionalconstraints.

In angiosperm systematics the application of noncod-

ing cpDNA sequences to low-level phylogenetic studies

is now routine (e.g., Shaw et al., 2005; and references

therein). A large number of diVerent noncoding regions

of the chloroplast genome have been investigated in

angiosperms, some of which are highly variable while

others show relatively little variation (Shaw et al., 2005).

These investigations have been facilitated by the large

* Corresponding author. Fax: +1 865 974 2258.

E-mail address:[email protected] (R.L. Small).
mailto:%[email protected]:%[email protected]:%[email protected]


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510 R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509522

number of complete chloroplast genome sequences that

are available from a wide phylogenetic array of angio-

sperms. The availability of these genome sequences has

provided the opportunity to develop universal angio-

sperm PCR primers in conserved coding regions that

Xank the more variable noncoding regions.

Molecular systematic studies in lycophytes andmonilophytes (leptosporangiate ferns, eusporangiate

ferns, Psilotaceae, and Equisetaceae; see Pryer et al.,

2004) have generally relied on a subset of the sequences

used in angiosperm systematics. The gene rbcL has been

used extensively in studies for both higher-level and

lower-level taxa (Dubuisson, 1997; Dubuisson et al.,

1998, 2003; Gastony and Johnson, 2001; Geiger and

Ranker, 2005; Hasebe et al., 1993, 1994, 1995; HauXer

and Ranker, 1995; HauXer et al., 1995, 2003; Hauk,

1995; Hauk et al., 2003; Hennequin et al., 2003; Kato

and Setoguchi, 1998; Korall and Kenrick, 2002, 2004;

Little and Barrington, 2003; Murakami et al., 1999; Nak-

azato and Gastony, 2003; Pinter et al., 2002; Pryer, 1999;

Pryer et al., 2001a,b, 2004, 1995; Ranker et al., 2003,

2004; Sano et al., 2000; Schneider et al., 2002, 2004a,c;

Skog et al., 2004; Wolf, 1995; Wolf et al., 1999). Other

genes such as atpB (Pryer et al., 2001a, 2004; Ranker

et al., 2003, 2004; Wolf, 1997) and rpS4 (Guillon, 2004;

Hennequin et al., 2003; Pryer et al., 2001a, 2004; San-

chez-Baracaldo, 2004a,b; Schneider et al., 2002, 2004c;

Smith and CranWll, 2002) have also been employed.

Among noncoding cpDNA regions, relatively few have

been used in lycophyte and monilophyte studies with the

trnLUAAtrnFGAA intergenic spacer (Taberlet et al.,

1991) being the most widely used by far (Eastwood et al.,2004; Geiger and Ranker, 2005; HauXer et al., 2003;

Hauk et al., 2003; Pinter et al., 2002; Ranker et al., 2003;

Rouhan et al., 2004; Schneider et al., 2004a,c; Skog et al.,

2002, 2004; Smith and CranWll, 2002; Su et al., 2005; Van

den Heede et al., 2003; Wikstrom et al., 1999) as it is in

angiosperms (Shaw et al., 2005). The trnSGGArpS4

intergenic spacer has also been used in a number of

recent studies (Guillon, 2004; Hennequin et al., 2003;

Perrie et al., 2003; Rouhan et al., 2004; Sanchez-Bara-

caldo, 2004a,b; Schneider et al., 2004b,c; Skog et al.,

2004; Smith and CranWll, 2002). The relatively rare use

of noncoding regions in lycophyte and monilophyte sys-

tematics is due in part to the necessary reliance on PCR

primers developed in angiosperm systematics. Unlike

angiosperms, only three complete chloroplast genomes

are available for lycophytes and monilophytes: Adian-

tum capillus-veneris (Wolf et al., 2003; GenBank Acces-

sion No. NC_004766), Huperzia lucidula (Wolf et al.,

2005; GenBank Accession No. AY660566), and Psilotum

nudum (Wakasugi et al., unpublished data, GenBank

Accession No. NC_003386). Despite the availability of

potential primers for numerous regions, many of the

PCR primers published for angiosperm studies may not

work in lycophytes or monilophytes due either to

sequence diVerences in the primer binding sites or rear-

rangements of the chloroplast genome.

Shaw et al. (2005)evaluated the ampliWcation and phy-

logenetic utility of 21 diVerent noncoding cpDNA regions

in a wide range of seed plant lineages. The purpose of the

present study was to evaluate the potential applicability of

these regions in lycophytes and monilophytes. To that endwe surveyed 30 species that represent the phylogenetic

breadth of lycophyte and monilophyte lineages (Hasebe

et al., 1995; Pryer et al., 1995, 2001a, 2004). Using these

exemplars we determined whether or not a subset of the

PCR primers used in the Shaw et al. (2005) study would

work in lycophytes and monilophytes. Several additional

regions were surveyed that were not included in the Shaw

et al. (2005) study, and in some cases new primers were

developed speciWcally for lycophytes and monilophytes.

Finally, to evaluate the relative phylogenetic utility of

some of these regions we ampliWed and sequenced three

cpDNA regions for members ofBotrychium s.l. and Hel-

minthostachys (Ophioglossaceae). Previous phylogenetic

studies in Ophioglossaceae have employed data from

rbcL and the trnLUAAtrnFGAA intergenic spacer (Hauk

et al., 2003). To complement these data and assess rela-

tive phylogenetic utility of diVerent regions we ampliWed

and sequenced two cpDNA regions that are particularly

useful in seed plants: the trnSGCUtrnGUUC intergenic

spacer+ the trnGUUC intron (hereafter trnStrnGtrnG);

and the rpL16 intron. In addition we ampliWed and

sequenced the trnSGGArpS4 intergenic spacer+ rpS4

gene because it has become widely employed in monilo-

phyte molecular systematics (Guillon, 2004; Hennequin

et al., 2003; Perrie et al., 2003; Rouhan et al., 2004; San-chez-Baracaldo, 2004a,b; Schneider et al., 2004b,c; Skog

et al., 2004; Smith and CranWll, 2002).

2. Materials and methods

2.1. Plant materials

Thirty species representing a broad phylogenetic

range of lycophyte and monilophyte lineages were

included in the study (Table 1). These included represen-

tatives of all three lycophyte families (Isotaceae, Lyco-podiaceae, and Selaginellaceae) as well as a range of

monilophyte families (eusporangiate ferns including Psi-

lotaceae and Equisetaceae, and leptosporangiate ferns).

Materials were either from Weld collections or green-

house grown plants. DNAs of Cyatheaceae species were

provided by D. Conant (Lyndon State College, VT).

Species of Ophioglossaceae chosen for detailed analysis

represent Helminthostachys and Botrychium s.l., the lat-

ter now segregated into Botrychium s.s., Sceptridium, and

Botrypus (Hauk et al., 2003; Table 1). Based on the

Ophioglossaceae phylogeny of Hauk et al. (2003)

Helminthostachys zeylanica was chosen as the outgroup.


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R.L. Small et al. / Molecular Phylogenetics and Evolution 36 (2005) 509522 511

2.2. Molecular methods

DNAs obtained speciWcally for this study were

extracted from leaf material (stem material from Equise-

tum and Psilotum) using the Plant DNeasy Mini Kit

(Qiagen); DNA extraction and PCR ampliWcation pro-

tocols for Ophioglossaceae were previously described by

Hauk et al. (2003). PCR ampliWcation was performed in

25L reactions with the following components: 1L

total genomic DNA (10100 ng), 1 PCR buVer (Pan-

Vera/TaKaRa), 200M each dNTP, 3.0 mM MgCl2(except for trnStrnGtrnGwhich used 1.5 mM MgCl2),

0.2g/L bovine serum albumin, 0.1 mM each primer,

and 0.625U rTaq DNA polymerase (PanVera/TaKaRa).

PCR ampliWcation primers are described in Table 2. All

PCR experiments included a negative control (no DNA)

reaction to monitor for contamination. Most regions

were PCR ampliWed using the following cycling condi-

tions: 30 cycles of 95C 1 min, 50C 1 min followed by a

slow ramp (1C/8 s) to 65 C, 65 C 4min. The trnS

trnGtrnG region was ampliWed using a 2-step PCR

cycling protocol: 30 cycles of 94C 1 min, 66C 4 min.

The trnTtrnL spacer, trnL intron, trnLtrnFspacer, and

rpS16regions were ampliWed using the following cycling

Ta le 1

Lycophyte and monilophyte taxa sampled for cpDNA ampliWcation, and Ophioglossaceae (Botrychium s.l. and Helminthostachys) species sampled

for DNA sequencing

Numbers correspond to lanes in Fig. 2. Voucher specimens are deposited at the University of Tennessee Herbarium (TENN) unless otherwise noted.

Family Taxon Source Voucher

Lycophytes

1 Lycopodiaceae Huperzia lucidula Carter Co., Tennessee, USA R. Small 162

2 Selaginellaceae Selaginella arenicola Lake Co., Florida, USA J. Beck 6004

3 Isotaceae Isotes Xaccida Wakulla Co., Florida, USA R. Small 296

Eusporangiate Ferns

4 Equisetaceae Equisetum sp. Greenhouse R. Small 284

5 Psilotaceae Psilotum nudum Greenhouse R. Small 285

6 Ophioglossaceae Ophioglossum vulgatum Greenhouse R. Small 286

7 Marattiaceae Angiopteris evecta Greenhouse R. Small 287

Leptosporangiate Ferns

8 Osmundaceae Osmunda cinnamomea Graham Co., North Carolina, USA E. Lickey 0330

9 Hymenophyllaceae Trichomanes petersii Graham Co., North Carolina, USA E. Lickey 0327

10 Schizaeaceae Lygodium japonicum Greenhouse R. Small 288

11 Marsileaceae Marsilea quadrifolia Greenhouse R. Small 289

12 Salviniaceae Salvinia sp. Greenhouse R. Small 290

13 Cyatheaceae Cnemidaria horrida D. Conant 4859

14 Cyatheaceae Cyathea arborea D. Conant 4822

15 Pteridaceae Adiantum pedatum Graham Co., North Carolina, USA E. Lickey 0325

16 Pteridaceae Cheilanthes lanosa Blount Co., Tennessee, USA E. Lickey 0322

17 Pteridaceae Pellaea atropurpurea Knox Co., Tennessee, USA R. Small 295

18 Pteridaceae Ceratopteris richardii Greenhouse R. Small 291

19 Dennstaedtiaceae Dennstaedtia punctilobula Blount Co., Tennessee, USA E. Lickey 0324

20 Aspleniaceae Asplenium platyneuron Blount Co., Tennessee, USA R. Small 283

21 Woodsiaceae Cystopteris protrusa Graham Co., North Carolina, USA E. Lickey 0328

22 Woodsiaceae Onoclea sensibilis Sevier Co., Tennessee, USA E. Lickey 0333

23 Woodsiaceae Deparia achrostichoides Graham Co., North Carolina, USA E. Lickey 0322

24 Woodsiaceae Athyrium felixfemina Graham Co., North Carolina, USA E. Lickey 0331

25 Dryopteridaceae Dryopteris marginalis Graham Co., North Carolina, USA E. Lickey 0326

26 Dryopteridaceae Cyrtomium sp. Greenhouse R. Small 292

27 Dryopteridaceae Polystichum acrostichoides Sevier Co., Tennessee, USA E. Lickey 0334

28 Davalliaceae Nephrolepis sp. Greenhouse R. Small 293

29 Davalliaceae Davallia sp. Greenhouse R. Small 29430 Polypodiaceae Polypodium appalachianum Graham Co., North Carolina, USA E. Lickey 0329

Ophioglossaceae Botrychium campestre Iowa, USA Farrar s.n., ISC

Botrychium simplex Mt. Ashland, Oregon, USA Hauk 619, NCU

Botrychium lunaria Marathon, Ontario, Canada Hauk 564, NCU

Botrychium lanceolatum Chippewa Co., Michigan, USA Hauk 571, NCU

Sceptridium dissectum Chapel Hill, North Carolina, USA Hauk 621, NCU

Sceptridium japonicum Japan Sahashi s.n., TOHO, DEN

Sceptridium lunarioides Dale Co., Alabama, USA Watkins 29, ISC

Botrypus virginianus Alger Co., Michigan, USA Hauk 575, NCU

Botrypus strictus Japan Sahashi s.n., TOHO, DEN

Helminthostachys zeylanica Japan Sahashi s.n., TOHO, DEN


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conditions: 30 cycles of 94C 1min, 50 C 1min, 72 C

2min.

The chloroplast regions we screened (Table 2) include

both introns and intergenic spacers. The speciW

c regionswe chose to screen were based on (1) the results of studies

of seed plant cpDNA (Shaw et al., 2005); (2) reference to

the literature for noncoding cpDNA regions used in previ-

ous lycophyte and monilophyte studies; and (3) examina-

tion of the noncoding regions found in the completely

sequenced Adiantum and Psilotum chloroplast genomes.

These regions are all found in the large single copy (LSC)

region of angiosperm chloroplast genomes, and most are

also found in the LSC of the Adiantum and Psilotum chlo-

roplast genomes as shown in Fig. 1. Some rearrangements

(e.g., inversions, translocations) of monilophyte chloro-

plast genomes relative to angiosperm chloroplast genomes

are apparent upon comparison of the cpDNA genome

maps ofPsilotum and Adiantum with typical angiosperms

such as Nicotiana (Wakasugi et al., 1998) (Fig. 1). Further,

some genes are present in some species chloroplastgenomes, but not in others (Fig. 1). Most primers used in

this study (Table 2) were previously described from angio-

sperm studies. A few primer sets, however, were designed

speciWcally for this study (atpF, ycf3, trnPpetG, trnM

trnV, rpL16, rpS16; see Table 2).

For sequencing of trnStrnGtrnG, trnSrpS4, and

some rpL16 in Ophioglossaceae (Botrypus virginianus,

B. strictus, Sceptridium japonicum, and Helminthostachys

zeylanica), PCR products were cleaned prior to sequenc-

ing using the ExoSAP-IT kit (United States Biochemi-

cal). PuriWed PCR products were sequenced with the ABI

Prism Big Dye Terminator cycle sequencing kit v. 3.1 and

Ta le 2

Chloroplast DNA regions ampliWed with primers

a Internal primers used for sequencing the trnStrnGregion in Ophioglossaceae only.b rpL16primers used for ampliWcation and sequencing ofBotrychium s.s. and S. dissectum.

Region Primers Reference

sbAtrnHGUG spacer trnHGUG: CGC GCA TGG TGG ATT CAC AAT CC Tate and Simpson (2003)

psbA: GTT ATG CAT GAA CGT AAT GCT C Sang et al. (1997)

trnKUUU intron/matKgene trnK-3914F: TGG GTT GCT AAC TCA ATG G Johnson and Soltis (1994)

trnK-2R: AAC TAG TCG GAT GGA GTA G Johnson and Soltis (1994)

rpS16intron rpS16-F-fern: AAR CGR TRT GGT AGR AAG CAA This paper

rpS16-R-fern: CGR GAT TGR RCA TCA ATT GCA A This paper

trnSGCUtrnGUUC spacer + intron trnSGCU: AGA TAG GGA TTC GAA CCC TCG GT Shaw et al. (2005)

3 trnGUUC: GTA GCG GGA ATC GAA CCC GCA TC Shaw et al. (2005)atrnG5 2G: GCG GGT ATA GTT TAG TGG TAA AA Shaw et al. (2005)atrnG5 2S: TTT TAC CAC TAA ACT ATA CCC GC Shaw et al. (2005)

atpFintron atpF-F: TAT YTT GGA RAG GGA GTG T This paper

atpF-R-fern: TTA RGY TTA TCA GTA GCT TCT This paper

trnCGCApsbMspacer trnCGCAF: CCA GTT CRA ATC YGG GTG Shaw et al. (2005)

psbMR: ATG GAA GTA AAT ATT CTY GCA TTT ATT GCT Shaw et al. (2005)

sbMtrnDGUC spacer psbMF: AGC AAT AAA TGC RAG AAT ATT TAC TTC CAT Shaw et al. (2005)

trnDGUCR: GGG ATT GTA GYT CAA TTG GT Shaw et al. (2005)

trnCGCArpoBspacer rpoB: CKA CAA AAY CCY TCR AAT TG Shaw et al. (2005)

trnCGCAF: CCA GTT CRA ATC YGG GTG Shaw et al. (2005)

ycf3 introns ycf3.x1.F: GCW TTT ACY TAT TAY AGA GAT G This paper

ycf3.x3.R: TNG AAT GGC CTG TTC TCC This paper

trnSGGArpS4 spacer + gene trnSGGA: TTA CCG AGG GTT CGA ATC CCT C Shaw et al. (2005)

rps4.5: ATG TCS CGT TAY CGA GGA CCT Souza-Chies et al. (1997)

trnTUGUtrnLUAA spacer a2: CAA ATG CGA TGC TCT AAC CT Cronn et al. (2002)

b: TCT ACC GAT TTC GCC ATA TC Taberlet et al. (1991)

trnLUAA intron c: CGA AAT CGG TAG ACG CTA CG Taberlet et al. (1991)

d: GGG GAT AGA GGG ACT TGA AC Taberlet et al. (1991)

trnLUAAtrnFGAA spacer e: GGT TCA AGT CCC TCT ATC CC Taberlet et al. (1991)

f: ATT TGA ACT GGT GAC ACG AG Taberlet et al. (1991)

trnVUACtrnMCAU intron + spacer trnVUAC: GGC TAT ACG GRY TYG AAC CGT A This paper

trnMCAU: CCT ACT ATT GGA TTY GAA CCA ATG ACT C This paper

trnPUGGpetGspacer trnPUGG: TGT AGC GCA GCY YGG TAG CG This paper

petG2: CAA TAY CGA CGK GGY GAT CAA TT This paper

rpL20-rpS12 spacer 5 rpS12: ATT AGA AAN RCA AGA CAG CCA AT Shaw et al. (2005)

rpL20: CGY YAY CGA GCT ATA TAT CC Shaw et al. (2005)sbB-psbHspacer psbB: TCC AAA AAN KKG GAG ATC CAA C Shaw et al. (2005)

psbH: TCA AYR GTY TGT GTA GCC AT Shaw et al. (2005)

rpL16intron rpL16-F-fern: ATG CTT AGT GTG YGA CTC GTT This paper

rpL16-R-fern: TCC SCN ATG TTG YTT ACG AAA T This paperb8R: GCT ATG CTT AGT GTG TGA CTC Asmussen (1999)b1067F: CTT CCT CTA TGT TGT TTA CG Asmussen (1999)


5/14


run on an ABI Prism 3100 automated sequencer (Univer-

sity of Tennessee Molecular Biology Resource Facility).

Sequencing electropherograms were assembled and

edited using Sequencher 4.1.2 (GeneCodes). The rpL16

sequences of Botrychium s.s., and Sceptridium dissectum

were cloned using the Qiagen PCR Cloning kit according

to the manufacturers recommendations (Valencia, CA).

The rpL16 sequence of S. lunarioides was sequenced

directly from ampliWed product puriWed using a QIA-

quick PCR PuriWcation kit (Valencia, CA). These tem-

plates were sequenced with the ABI Prism BigDye

Terminator Cycle Sequencing Reaction Kit and run on

an ABI 373XL Stretch DNA sequencer.

2.3. Analyses

To evaluate the ampliWcation success of each of the 18

noncoding regions in the 30 lycophyte and monilophyte

lineages, PCR ampliWcation products were run on 1.5%

agarose gels and digitally documented. A subset of the

PCR products was sequenced to conWrm their identity.

For each cpDNA region that was successfully ampliWed

three of the PCR products were sequenced. In most cases

PCR products from one lycophyte, one eusporangiate

fern, and one leptosporangiate fern were sequenced.

To assess the utility of the trnStrnGtrnG, trnS

rpS4, and rpL16 regions in Botrychium s.l. relative to

Fig. 1. Comparative maps of the Large Single Copy (LSC) region of the two completely sequenced monilophyte chloroplast genomes ( Psilotum and

Adiantum) relative to a typical angiosperm (Nicotiana) chloroplast genome. Gene acronyms are shown in order from the top (junction of Inverted

Repeat B and LSC) to bottom (junction of LSC and Inverted Repeat A) only to show gene order and presenceno indication of size of regions is

inferred. Noncoding cpDNA regions ampliWed for this study are shown as black boxes on the Nicotiana map. DiVerences between gene arrangement

or presence/absence are shown on the map and are indicated by letter: (A) no genes exist between accD and rbcL in Nicotiana, but a trnRCCG gene is

found here in Psilotum and a trnSeCUCA gene (coding for the modiWed amino acid selenocysteine) in Adiantum. (B) A trnTUGU gene is found here in

both Psilotum and Nicotiana, but is missing in Adiantum. (C) An inversion of the trnTGGUpsbDpbsCtrnSUGAycf9trnGGCC region is present in

both Adiantum and Psilotum relative to Nicotiana . (D) An inversion and translocation of the trnCGCAycf6psbMregion is found in Adiantum rela-

tive to Nicotiana and Psilotum. (E) The trnDGUC gene has been translocated in Adiantum relative to Nicotiana and Psilotum. (F) Theycf12 gene is

present in Psilotum and Adiantum, but missing in Nicotiana. (G) ThepsaMgene is present in Psilotum, but missing in Adiantum and Nicotiana . (H)

The trnSCGA gene is present in Psilotum, but missing in Adiantum and Nicotiana . (I) The chlBgene is present in Adiantum, but missing in Psilotum and

Nicotiana. (J) The rpS16gene is present in Adiantum and Nicotiana, but missing in Psilotum. (K) The trnKUUU gene plus the matK gene which is

encoded in the trnKintron are present in both Psilotum and Nicotiana, but the trnKexons are missing in Adiantum. (L) ThepsbAtrnHGUG region is

present in all three chloroplast genomes, but has been translocated into the inverted repeat in Adiantum.


6/14


available data from rbcL and trnLtrnF a number of

diVerent approaches were used. First, for each data set

descriptive statistics were calculated (sequence length,

number and percentage of variable characters, number

and percentage of phylogenetically informative charac-

ters). In addition, phylogenetic analyses of each data set

were performed individually to compare levels of resolu-tion and support (branch lengths, bootstrap and decay

values, consistency and retention indices).

Sequences were initially aligned using Clustal_X

(Thompson et al., 1997), and alignments were manually

reWned in MacClade 4.0 (Maddison and Maddison,

2000). For phylogenetic analysis gaps in the alignment

were treated as missing data, but the individual gaps

were subsequently coded as binary characters and

added to the end of the sequence matrix. Phylogenetic

analyses were performed using the optimality criterion

of maximum parsimony in PAUP* 4.0b10 (SwoVord,

2002). Exhaustive searches were conducted to Wnd all

maximally parsimonious trees, bootstrap support was

estimated using 1000 bootstrap replicates with branch

and bound searches, and decay analyses were conducted

with a reverse-constraints approach as implemented in

TreeRot v. 2 (Sorenson, 1999). One 52 bp region of the

rpL16data set that consisted almost entirely of varying

lengths of runs of A and G nucleotides was excluded

from phylogenetic analysis due to ambiguous

alignment.

3. Results

3.1. AmpliWcation of noncoding cpDNA in lycophytes and

monilophytes

Eighteen primer sets (Table 2) were screened for their

ability to amplify noncoding cpDNA regions in 30 lyco-

phyte and monilophyte species (Table 1). Of those 18

primer sets screened, eight primer sets showed good

ampliWcation (a single strong band) in most species. Six

other primer sets showed good ampliWcation in a subset

of species screened. Finally, four primer sets produced

either no ampliWcation products or resulted in the ampli-

W

cation of multiple weak products or smears. Fig. 2shows representative gel pictures for those regions that

ampliWed in at least some species. This information is

summarized in Fig. 3.

One particular taxon (Selaginella) was problematic in

these ampliWcation experiments. Despite trying ampliW-

cation from DNA of three diVerent Selaginella species

(S. apoda, S. arenicola, and S. kraussiana) we consis-

tently had diYculty getting good ampliWcation from

Selaginella even for those cpDNA regions that worked

in all other species tested (see lane 2 ofFig. 2).

To conWrm that the target region was ampliWed using

these PCR primers and conditions we sequenced a sub-

set of the ampliWcation products and used BLAST

(Altschul et al., 1990) to search GenBank for matching

sequences. In all cases the sequenced PCR product

matched sequences in GenBank from the appropriate

cpDNA region.

It should be noted that the PCR conditions used in

these ampliWcation experiments were those we havefound to be generally useful across a wide range of tem-

plates and primers. Given the large number of taxa and

cpDNA regions, we did not attempt to optimize reaction

conditions for each region. It is apparent from evalua-

tion ofFig. 2 that in some cases multiple PCR products

were ampliWed or ampliWcation was weak in some taxa.

Further optimization of PCR conditions (e.g., annealing

temperature, MgCl2 concentration) would likely

improve the ampliWcation of those regions. Additionally,

several region-speciWc issues also became apparent dur-

ing the course of this investigation and are discussed in

the following paragraphs.

The trnKUUU intron/matKgene region is widely used

in seed plant systematics, but did not amplify in our

experiments. As discussed by Wolf et al. (2003), while

the matKgene is present in Adiantum, a large inversion

(Hasebe and Iwatsuki, 1990) has an endpoint near

matK and no trnK exons have been detected in

Adiantum.

The trnCGCArpoBregion in Adiantum has undergone

a small inversion relative to its orientation in angio-

sperm chloroplast DNA (Fig. 1). As a result, the trnCGCA

gene is in a reverse orientation in Adiantum relative to

angiosperms. To account for this in our ampliWcation

experiments we used a primer on the opposite strand oftrnCGCA relative to the primer usually used in angio-

sperms (see e.g., Shaw et al., 2005).

The trnL intron and trnL-Fintergenic spacer has been

used in a previous phylogenetic study in Huperzia (Wik-

strom et al., 1999). The length of the trnL intron + trnL-F

spacer reported by Wikstrom et al. (1999) from H. luci-

dula, however, is signiWcantly shorter than the size of the

corresponding PCR products obtained in this study. The

combined trnL intron+ trnL-F spacer sequence (Gen-

Bank Accession No. AJ224591) used by Wikstrom et al.

(1999) is 833bp. In our ampliWcation experiments the

trnLintron from

H. lucidulais ca. 500 bp (which agrees

with the GenBank accession), but the trnL-Fspacer is ca.

1500 bp (Fig. 2). This apparent discrepancy is due to the

use of only a partial sequence by Wikstrom et al. (1999;

and N. Wikstrom, pers. comm.). Further, the size of these

regions in the complete chloroplast genome sequence for

H. lucidula (Wolf et al., 2005; GenBank Accession No.

AY660566) is consistent with our results.

3.2. Phylogeny of Botrychium s.l.

Sequences of the trnStrnG intergenic spacer+ the

trnG intron, the rpL16 intron, and the trnSrpS4


7/14


Fig. 2. Gel photos showing the ampliWcation success of the noncoding cpDNA regions tested in 30 lycophyte and monilophyte species. Only those

regions in which ampliWcation for at least some species was successful are shown. Lane numbers are the same across all photos and match the num-

bers given in Table 1. In each gel photo a molecular weight marker is shown at each end and in the middle [band sizes in decreasing order: 2.68, 2.0,

1.5, 1.2, 1.0 kb (brighter band), 0.90.1 kb in 0.1 kb increments].


8/14


spacer+ rpS4 gene were obtained for nine species of

Botrychium s.l. and the outgroup Helminthostachys

zeylanica. These newly generated sequences have been

deposited in GenBank (Accession Nos. AY870407

AY870436). The species chosen for this analysis (Table

1) are a subset of the species included in larger analyses

of the family (Hauk et al., 2003) and represent all of the

major botrychioid clades recovered in those analyses.

Phylogenetic analyses of the three new sequence data

sets (rpL16, trnSrpS4, trnStrnGtrnG) and the equiva-

lent data sets from the previously published analyses

(rbcL, trnLtrnF) were performed independently. Phylo-

genetic analyses recovered a single most parsimonious

tree from each data set except for rpL16 from which

three equally parsimonious trees were recovered. All

data sets recovered an identical topology (Fig. 4) with

the exception of the B. simplex/B. lunaria/B. campestre

clade. Two of the data sets (rbcL, trnLtrnF) found a

topology of (B. lunaria (B. simplex, B. campestre)); two

of the data sets (trnStrnGtrnG, trnSrpS4) found a

topology of (B. campestre (B. lunaria, B. simplex)); the

strict consensus tree of the three trees recovered in the

rpL16analysis had a polytomy with relationships among

these three species unresolved. The strict consensus tree

resulting from comparison of trees recovered from the

independent data sets is shown in Fig. 4, as are the sup-

port measures for each node from the diVerent data sets

(character state changes, bootstrap values, decay values

for each node).

Data set characteristics (sequence length, number of

variable and parsimony-informative nucleotide substitu-

tions and indels, consistency index, retention index, and

tree length) are described in Table 3. While Table 3

shows each noncoding region separately for comparison

(e.g., trnStrnGspacer and trnGintron; trnSrpS4 spacer

and rpS4 gene) as well as combined into ampliWed units

(e.g., trnStrnG spacer+ trnG intron; trnSrpS4

spacer+ rpS4 gene) the following descriptions focus on

Fig. 3. Summary of ampliWcation success of the 18 noncoding cpDNA regions tested in 30 lycophyte and monilophyte taxa. Black boxes indicate a

single strong band ampliWed for this region from this species. Grey boxes indicate that a weak band ampliWed, or that multiple bands ampliWed for

this region from this species. Blank boxes indicate that no ampliWcation product was observed for this region from this taxon.


9/14


the combined data sets because these were used for the

phylogenetic analyses. Consistency and retention indi-

ces are generally similar across data sets, ranging from0.830.88 to 0.730.81, respectively. The data sets vary

widely in size (aligned length) with trnLtrnFbeing the

smallest (369 nt) and trnStrnGtrnG being the largest

(1830 nt). Numbers and percentages of variable and par-

simony-informative sites also varied considerably across

data sets. The lowest numbers and percentages of both

variable and parsimony-informative sites were obtained

with rbcL, as expected given the conserved nature of this

gene. Among the other sequenced regions the trnLtrnF

intergenic spacer provided the highestpercentage of var-

iable (46.9%) and parsimony-informative (16.3%) sites,

while at the same time providing the lowest overall num-

bers of variable (173) and parsimony-informative (60)

sites. The trnStrnGtrnG region provided the greatest

number of both variable (458) and parsimony-informative(181) sites, with percentages similar to the other regions.

As expected, the number of variable and parsimony-

informative sites in a given data set is associated with the

overall sequence length of the data set. In an analysis of

cpDNA sequence variation in seed plants Shaw et al.

(2005) showed that sequence length accounted for any-

where from 22 to 83% of the variation in the number of

variable characters observed in a data set. To assess the

relationship between sequence length and the number of

variable and parsimony-informative characters in our

Botrychium s.l.+ Helminthostachys data sets we

regressed sequence length by number of both variable

Fig. 4. Consensus phylogenetic tree from analyses of sequence data from Wve cpDNA regions for Botrychium s.l. + Helminthostachys . Relative mea-

sures of support (s, steps; b, bootstrap; d, decay) for each of the numbered nodes are shown for each of the Wve data sets.

Ta le 3

Characteristics of the Wve cpDNA sequence data sets for Botrychium s.l.

The trnSrpS4 spacer + gene and trnStrnGspacer + trnGintron data sets were each analyzed together, but are shown both separated into individual

units and together here for comparison.

Data set Aligned sequence

length (range)

nucleotides

Number (%)

variable

nucleotide

substitutions

Number (%)

informative

nucleotide

substitutions

Number of

indels

(informative

indels)

Consistency

index/retention

index

Tree

length

rbcL gene 1330 (13211330) 158 (11.9%) 58 (4.4%) 0 (0) 0.87/0.76 191

trnLtrnFspacer 369 (305368) 173 (46.9%) 60 (16.3%) 19 (5) 0.85/0.74 227rpL16intron 791 (726747) 227 (28.7%) 81 (10.2%) 29 (2) 0.88/0.81 282

trnSrpS4 spacer + gene 956 (938-949) 246 (25.7%) 77 (8.1%) 11 (1) 0.88/0.77 297

trnSrpS4 spacer 379 (360372) 139 (36.6%) 40 (10.6%) 11(1) 0.86/0.70 177

rpS4 gene 577 (577577) 107 (18.5%) 37 (6.4%) 0 (0) 0.92/0.85 120

trnStrnGspacer + intron 1830 (16991771) 458 (25.0%) 181 (9.9%) 38 (6) 0.83/0.73 597

trnStrnGspacer 1047 (924991) 278 (26.6%) 119 (11.4%) 28 (4) 0.81/0.72 371

trnGintron 760 (749757) 180 (23.7%) 62 (8.2%) 10 (2) 0.86/0.74 227


10/14


and parsimony-informative sites for the noncoding

regions sequenced (Fig. 5). For this analysis each region

was separated into individual noncoding regions (trnL

trnFspacer, trnSrpS4 spacer, rpL16intron, trnGintron,

and trnStrnGspacer). This analysis indicates that 81%of the variation in the number of variable sites and 79%

of the variation in the number of parsimony-informative

sites is explained by sequence length. Equivalent data for

the genes rbcL and rpS4 are also shown in Fig. 5,

although these data were not included in the regression

analyses.

4. Discussion

4.1. AmpliWcation of noncoding cpDNA in lycophtes and

monilophytes

Most lycophyte and monilophyte molecular phyloge-

netic studies have relied on a small number of cpDNA

sequences, namely the gene rbcL, the trnLtrnF inter-

genic spacer, and the trnSrpS4 intergenic spacer+ rpS4

gene. In many cases, these data sets have provided suY-

cient phylogenetic resolution, while in other cases, espe-

cially in studies of very closely related species or

intraspeciWc variation, insuYcient resolution is obtained

due to a paucity of phylogenetically informative charac-

ters. This situation is similar to angiosperm studies

where a few popular regions are predominantly used. A

recent study in seed plants (Shaw et al., 2005) demon-

strated that several rarely used cpDNA regions were

generally much more variable than the widely used

regions. The present study was undertaken to assess the

potential applicability of some of these same regions in

lycophyte and monilophyte studies.

The PCR-ampliWcation experiments shown in Fig. 2and summarized in Fig. 3 demonstrate that a wide vari-

ety of cpDNA regions can be ampliWed in a broad range

of lycophytes and monilophytes. Eight regions ampliWed

universally or nearly universally (psbAtrnH, trnStrnG

trnG, trnSrpS4, trnL, trnLtrnF, trnMtrnV, trnPpetG,

and rpL16). Six other regions ampliWed well in a subset

of taxa (rpS16, atpF, trnCrpoB, psbMtrnC, trnD

psbM, and ycf3). Finally, four regions ampliWed poorly

or not at all from most taxa (trnK/matK, psbBpsbH,

rps12rpL20, and trnTtrnL).

4.2. Relative phylogenetic utility ofWve data sets in

Botrychium s.l.

Tso test the relative phylogenetic utility of diVerent

cpDNA sequences in resolving relationships, we

analyzed representative species of Botrychium

s.l.+ Helminthostachys. Previously published work

(Hauk et al., 2003) used rbcL and trnLtrnFsequences to

address relationships in a larger analysis of Ophioglossa-

ceae. Both of these data sets provided similar and com-

patible resolution of relationships although support for

clades varied between data sets. To complement and

compare these published data sets we generated data for

nine species of Botrychium s.l.+ Helminthostachys fromthree additional cpDNA regions: the rpL16 intron, the

trnSrpS4 intergenic spacer+ rpS4 gene, and the trnS

trnGintergenic spacer+ trnGintron. With the exception

of the Botrychium s.s. clade, phylogenetic resolution was

comparable across all data sets (Fig. 4).

Relative levels of support, on the other hand, as mea-

sured by branch lengths, bootstrap values, and decay val-

ues varied widely between data sets (Fig. 4). Bootstrap

values were generally similar across data sets for those

nodes that are strongly supported in all data sets (e.g.,

nodes 1, 2, and 3 in Fig. 4). For those nodes that are rela-

tively weakly supported in some data sets, however,bootstrap values varied considerably. For example, node

6 in Fig. 4 (the S. japonicum + S. dissectum clade) has

bootstrap values of 58, 84, 74, 98, and 91% in rbcL, trnL

trnF, rpL16, trnSrpS4, and trnStrnGtrnG, respectively.

Branch lengths and decay values varied even more

widely among data sets than did bootstrap values. For

every node the trnStrnGtrnG data set provided the

longest branches (i.e., the most character support). Often

the diVerences in branch lengths are dramatic. For exam-

ple, node 3 in Fig. 4 has branch lengths of 10, 15, 18, 14,

and 42 in rbcL, trnLtrnF, rpL16, trnSrpS4, and trnS

trnGtrnG, respectively. Decay values follow a similar

Fig. 5. Scatter plot of sequence length vs. numbers of variable and par-

simony-informative characters for individual data sets. , indicates

parsimony-informative characters in noncoding regions; , indicates

variable characters in noncoding regions; , indicates parsimony-informative (PI) characters in the gene rpS4; , indicates variable

(var) characters found in the gene rpS4; , indicates parsimony-infor-

mative characters in rbcL; , indicates variable characters in rbcL. A

line of best Wt was calculated for sequence length vs. variable charac-

ters in the noncoding regions (upper line), and sequence length vs. par-

simony-informative characters in the noncoding regions (lower line).


11/14


pattern with node 3 having decay values of +10, +10,

+14, +10, and +33 in rbcL, trnL trnF, rpL16, trnSrpS4,

and trnStrnGtrnG, respectively.

Thus, with respect to the recovered topology all data

sets provide similar results and nearly complete resolu-

tion of most relationships. Comparisons of levels of sup-

port for the phylogeny, however, reveal diVerencesamong data sets and clearly show that some data sets

provide greater support for inferred relationships than

others. Overall, the trnStrnGtrnGdata set provides the

greatest character support, and generally the highest

bootstrap and decay values.

4.3. Relationship between sequence length and variation

There is, of course, an association between sequence

length and the number of phylogenetically informative

characters that a particular region can be expected to

provide. This association is borne out in an analysis of

sequence length vs. numbers of variable and phylogenet-

ically informative characters (Fig. 5). As sequence length

increases, the number of both variable and phylogeneti-

cally informative characters also increases with r2D 0.81

for variable characters and r2D0.79 for phylogenetically

informative characters. As expected, the genes rbcL and

rpS4 provide fewer variable or phylogenetically informa-

tive characters per unit of sequence compared to the

noncoding regions, presumably due to greater functional

constraints on these genes (Fig. 5).

Although a strong association exists between

sequence length and numbers of variable or phylogeneti-

cally informative characters, there remain diVerences inthe numbers of characters that are not accounted for by

sequence length alone (i.e., ca. 20% of the variation). A

portion of this variation is clearly stochastic due to our

Wnite sample size, but some of this variation may be due

to intrinsic diVerences in the phylogenetic utility of the

diVerent regions (see Shaw et al., 2005). These diVerences

in relative levels of variation may reXect the presence of

conserved elements within some noncoding regions such

promoter or regulatory motifs in intergenic spacers, or

conserved secondary structures in introns. Fig. 5 shows a

line of best Wt for both the variable and phylogenetically

informative characters. In the comparison of variablecharacters there are three data sets that lie above the line

of best Wt (i.e., have greater than predicted variable char-

acters per unit of sequence): the trnLtrnF spacer, the

rpL16 intron, and trnStrnG spacer. Two data sets lie

below the line and thus have lower than predicted vari-

able characters per unit of sequence: the trnSrpS4

spacer and the trnG intron. Further, there are pairs of

sequences with similar lengths, but relatively diVerent

numbers of variable characters. The trnLtrnFdata set

was 369 nt long with 173 variable characters while the

trnSrpS4 data set was 379 nt long, yet contained only

139 variable characters. In other words, the trnSrpS4

data set contained only 80% of the number of variable

characters found in the trnLtrnF data set despite the

fact that they are almost identical in length. Similarly,

the rpL16 intron and trnG intron were 791 and 760 nt

long, with 227 and 180 variable characters, respectively

(i.e., trnGhas 79% of the number of variable characters

of rpL16 despite similar lengths). A similar pattern isseen in the line of best Wt for sequence length vs. phyloge-

netically informative characters (Fig. 5).

Finally, it should be noted that the genes rbcL and

rpS4 both show considerably lower numbers of variable

and phylogenetically informative characters than the

noncoding regions of similar length (Fig. 5). An advan-

tage of using coding sequences is that they are trivial to

align relative to the sometimes challenging task of align-

ing noncoding regions. This advantage is clearly out-

weighed, however, by the lower numbers of variable

characters found in these regions, at least for analyses of

closely related species.

4.4. Choosing an appropriate region for analysis

The addition of the cpDNA noncoding regions

identiWed here to the arsenal of tools available to pterid-

ologists considerably expands the potential sources of

information available for phylogenetic inference. This

leads directly to the question of which particular region

or regions should be employed in any given study.

The analysis ofShaw et al. (2005) identiWed consider-

able variability in the amount of sequence variation

detected in diVerent noncoding cpDNA regions among

seed plants. In the study of Shaw et al. (2005) the ana-lyzed regions were grouped into tiers with tier 1

regions providing the greatest number of variable char-

acters, tier 2 regions providing fewer, and tier 3

regions providing the least. Based on these analyses it

was clear that the tier 1 regions should be explored Wrst

for any particular study as they are the most likely to

provide the greatest number of characters. It was also

noted, however, that no one cpDNA region was univer-

sally the most informative, and that considerable varia-

tion existed among plant lineages as to which cpDNA

region was the most informative. In other words, one

region may be the most informative in one lineage, whilea diVerent region may be the most informative in a

diVerent lineage. The analyses discussed above show that

among the regions surveyed here for Botrychium

s.l.+ Helminthostachys, the trnLtrnF spacer, the rpL16

intron, and the trnStrnG spacer provide greater than

predicted levels of variation while the trnSrpS4 spacer

and trnG intron provide lower than predicted levels of

variation. Comparative data to determine whether or

not this is generally true across lycophytes and monilo-

phytes are not yet available.

These observations lead to the conclusion that a pre-

liminary survey of several potential cpDNA regions in


12/14


the taxa of interest is a critical step in identifying which

cpDNA region or regions are likely to provide the most

variation in a given lineage (Shaw et al., 2005). Such a

preliminary study can be performed with as few as three

taxa where sequence data from numerous cpDNA

regions are generated from the three exemplars and rela-

tive levels of variation are compared across regions forthese three taxa (Shaw et al., 2005). The region or regions

showing the highest level of variation in this preliminary

survey are those most likely to also provide the greatest

number of phylogenetically informative sites in a

broader analysis (Shaw et al., 2005).

4.5. Conclusions

The data and analyses presented here show that numer-

ous cpDNA noncoding regions can be ampliWed in a wide

range of lycophytes and monilophytes, which expands the

number of potential sequences to choose from for phylo-

genetic studies in these lineages. Comparative sequence

analysis in Botrychium s.l.+Helminthostachys shows that

phylogenetic resolution is consistent among the Wve data

sets employed, but that levels of support for the inferred

phylogeny vary across data sets. In this particular exam-

ple, coding sequences such as the genes rbcL and rpS4,

provide relatively low levels of sequence variation per unit

of sequence compared to noncoding regions. Among the

noncoding regions sampled the trnLtrnF spacer, the

rpL16intron, and the trnStrnGintergenic spacer provide

greater levels of variability per unit of sequence than the

trnSrpS4 spacer and the trnG intron. These data, taken

together with the conclusions ofShaw et al. (2005), indi-cate that preliminary studies of the relative phylogenetic

utility of a given cpDNA region should be performed

prior to mounting a full-scale sequencing eVort using any

one region.

Acknowledgments

We thank Dave Conant (Lyndon State College) for

providing DNA of Cyatheaceae species; and the

National Science Foundation, the Hesler Fund from the

University of Tennessee Herbarium, and the DenisonUniversity Research Foundation for funding that sup-

ported this research. Paul Wolf and two anonymous

reviewers provided valuable feedback that improved the

manuscript.

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fern cpdna====one particular taxon selaginella was problematic

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